The present invention, in some embodiments thereof, relates to a nucleic acid expression construct and, more particularly, but not exclusively, to a nucleic acid construct for expression of α-galactosidase in plant cells, cells expressing the nucleic acid construct and the human α-galactosidase, and uses thereof.
Fabry disease is an X-linked lysosomal storage disease that is caused by deficient activity of lysosomal enzyme α-galactosidase A (α-Gal A). Patients with classic Fabry disease typically have α-Gal A activity of less than 1% and often demonstrate the full spectrum of symptoms, including severe pain in the extremities (acroparesthesias), hypohidrosis, corneal and lenticular changes, skin lesions (angiokeratoma), renal failure, cardiovascular disease, pulmonary failure, neurological symptoms and stroke. In atypical Fabry disease, individuals with residual enzyme activity demonstrate symptoms later in life, and the symptoms are usually limited to one or a few organs. Clinical manifestations in female carriers vary greatly because of random X-chromosome inactivation. Although carriers commonly remain asymptomatic throughout life, many demonstrate clinical symptoms as variable and severe as those of affected males. De Duve first suggested that replacement of the missing lysosomal enzyme with exogenous biologically active enzyme might be a viable approach to treatment of lysosomal storage diseases [Fed Proc. 23:1045 (1964)].
Recombinant α-Galactosidase A for enzyme replacement therapy has been produced in insect (sf9) cells (see U.S. Pat. No. 7,011,831) in human fibroblasts (see U.S. Pat. No. 6,395,884) and in plant cells (see U.S. Pat. No. 6,846,968 and WO2008/132743). Clinical trials with recombinant α-Gal A (agalsidase beta [Fabrazyme®]: Genzyme Corporation, Cambridge, Mass.; agalsidase alfa [Replagal®]: Shire Human Genetic Therapies Corporation, Cambridge, Mass.) have been performed, and both drugs have been approved for clinical use. As of 2009, more than 2000 patients had been treated with either one of the two available formulations, and in general, safety and efficacy of both have been demonstrated (Hoffmann, Orphanet J Rare Dis. 2009; 4:21).
Clinical experience has indicated that the available recombinant enzyme compositions differ with regard to patient tolerance, immunogenicity, dosage regimen and clinical efficacy (Hoffmann, Orphanet J Rare Dis. 2009; 4:21). Another drawback associated with the existing recombinant enzymes is their expense, which can place a heavy economic burden on health care systems. The high cost of these recombinant enzymes, when produced in mammalian cell cultures, bioreactors or insect cells results from a complex purification and modification protocol, and the relatively large amounts of the therapeutic required for existing treatments. There is therefore, an urgent need to reduce the cost of α-galactosidase so that this life saving therapy can be provided more affordably to all who require it.
Human lysosomal enzymes can be produced in transgenic plants in order to solve problems of safety, viral infections, immune reactions, production yield and cost. US 2002/0088024 teaches expression in plant cells of recombinant human α-galactosidase, using a rice-amylase ER targeting signal peptide, and a human α-galactosidase coding sequence truncated at the C-terminal portion for efficient expression and secretion into the intracellular fluid.
WO 97/10353 teaches production of lysosomal enzymes in plants, specifically IDUA and glucocerebrosidase, using the wound-induced MeGA promoter and FLAG tag for recovery. WO 97/10353 does not provide guidance for specific constructs expressing recombinant human α-galactosidase.
Cramer et al. (Curr. Topics Trans. 1999 Plants 95-118) reviews methods of expression of glucocerebrosidase and IDUA in plants in a similar manner as WO 97/10353, that is in the endomembrane (IF) of tobacco leaves, and describes plant expression in general but does not provide guidance for expression of human recombinant α-galactosidase.
US2006-0204487 teaches methods for the expression of human lysosomal enzymes in plants, employing a variety of strategies for targeting the recombinant polypeptides (specifically, glucocerebrosidase) for efficient glycosylation and glycan remodeling. Cloning and expression of recombinant human α-galactosidase from specific constructs is not disclosed.
WO2007/010533 teaches the administration of plant cells expressing recombinant bioactive molecules, for example, lysosomal enzymes such as human glucocerebrosidase and human α-galactosidase. Cloning and expression of recombinant human α-galactosidase is not disclosed.
WO2008/132743 describes cloning of a human α-galactosidase coding sequence into a plant expression vector, and its expression in tobacco cells, resulting in a biologically active, full length glycosylated α-galactosidase enzyme which demonstrated uptake in fibroblasts. However, more advanced α-galactosidase expression vectors for yet further increased efficiency of expression and production of recombinant human α-galactosidase protein having improved pharmacokinetics under clinical conditions are needed.